Rational incorporation of molecular adjuvants into a hybrid nanoparticle-based nicotine vaccine for immunotherapy against nicotine addiction

Abstract

Current clinically-tested nicotine vaccines have yet shown enhanced smoking cessation efficacy due to their low immunogenicity. Achieving a sufficiently high immunogenicity is a necessity for establishing a clinically-viable nicotine vaccine. This study aims to facilitate the immunogenicity of a hybrid nanoparticle-based nicotine vaccine by rationally incorporating toll-like receptor (TLR)-based adjuvants, including monophosphoryl lipid A (MPLA), Resiquimod (R848), CpG oligodeoxynucleotide 1826 (CpG ODN 1826), and their combinations. The nanoparticle-delivered model adjuvant was found to be taken up more efficiently by dendritic cells than the free counterpart. Nanovaccine particles were transported to endosomal compartments upon cellular internalization. The incorporation of single or dual TLR adjuvants not only considerably increased total anti-nicotine IgG titers but also significantly affected IgG subtype distribution in mice. Particularly, the nanovaccines carrying MPLA+R848 or MPLA+ODN 1826 generated a much higher anti-nicotine antibody titer than those carrying none or one adjuvant. Meanwhile, the anti-nicotine antibody elicited by the nanovaccine adjuvanted with MPLA+R848 had a significantly higher affinity than that elicited by the nanovaccine carrying MPLA+ODN 1826. Moreover, the incorporation of all the selected TLR adjuvants (except MPLA) reduced the brain nicotine levels in mice after nicotine challenge. Particularly, the nanovaccine with MPLA+R848 exhibited the best ability to reduce the level of nicotine entering the brain. Collectively, rational incorporation of TLR adjuvants could enhance the immunological efficacy of the hybrid nanoparticle-based nicotine vaccine, making it a promising next-generation immunotherapeutic candidate for treating nicotine addiction.

Keywords: Anti-nicotine antibody; Hybrid nanoparticle; Molecular adjuvant; Nicotine addiction; Nicotine vaccine; Toll-like receptors.

Figure 1

Characterization of NPs. (A) Schematic illustration and TEM images of liposomes, PLGA NPs, lipid-PLGA hybrid NPs, and adjuvant-loaded NanoNicVac particles. Scale bars represent 100 nm. (B) Average diameters of NanoNicVac particles. (C) Zeta-potential of NanoNicVac particles.

Figure 2

Cellular Uptake of NanoNicVac particles by dendritic cells. (A) Flow cytometry recorded events, (B) M.F.I. of CM-6, and (C) M.F.I. of AF647 of dendritic cells after being treated with free AF467-KLH+CM-6 (In free form) or NanoNicVac particles carrying AF647-KLH and CM-6 (In nanoparticles). AF647 was used to label KLH, and CM-6 was used as a model adjuvant that was loaded into the PLGA core. Significantly different: *, p < 0.05, ***, p < 0.001. (D) M.F.I. of NBD in dendritic cells after being treated with NanoNicVac particles loaded with different adjuvants. NBD was added to the lipid-layer to label NPs. (E) CLSM images of dendritic cells after being treated with NanoNicVac particles for 1, 2, or 4 h. AF647 was used as a model hapten to provide fluorescence and CM-6 was used as a model adjuvant loaded into the PLGA core. Scale bars represent 10 μm.

Figure 3

Immunogenicity of adjuvant-loaded NanoNicVac. The titers of anti-nicotine IgG antibodies elicited by NanoNicVac on (A) day 12, (B) day 26, and (C) day 40 were measured by ELISA. Significantly different compared to the previous studied date: & p < 0.05, && p < 0.01, and &&& p < 0.001. Significantly different compared to NanoNicVac group with no adjuvant: # p < 0.05, ## p < 0.01, and ### p < 0.001. Significantly different: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

Subtype distribution of anti-nicotine IgGs. The titers of anti-nicotine IgG subtypes on day 40, including (A) IgG1, (B) IgG2a, (C) IgG2b, and (D) IgG3, were assayed. (E) shows the relative percentages of subtype anti-nicotine IgGs. Significantly different compared to NanoNicVac with no adjuvant: # p < 0.05, ## p < 0.01, and ### p < 0.001. Significantly different: ** p < 0.01, *** p < 0.001.

Figure 5

Affinity of anti-nicotine antibodies elicited by NanoNicVac on (A) day 12, (B) day 26, and (C) day 40. The antibody affinity was estimated by competition ELISA. Significantly different compared to the IC₅₀ on day 12: & p < 0.05, && p < 0.01. Significantly different: * p < 0.05.

Figure 6

Pharmacokinetic efficacy of adjuvant-loaded NanoNicVac in mice. (A) Serum nicotine concentration. (B) Brain nicotine concentration. Mice were administered 0.06 mg/kg nicotine on day 42, and the brain and serum samples were collected 3 min after nicotine administration. Significantly different compared to NanoNicVac with no adjuvant: # p < 0.05, ## p < 0.01. Significantly different: * p < 0.05, ** p < 0.01.

Figure 7

Preliminary safety of adjuvant-loaded NanoNicVac particles. (A) Body weight of immunized mice. (B) Representative H&E staining images of major organs of mice immunized with NanoNicVac carrying different adjuvants.